REVIEW Open Access

Exploring the key communicator role ofexosomes in cancer microenvironmentthrough proteomicsHuiSu Kim1, Dong Wook Kim1 and Je-Yoel Cho1,2*

ABSTRACT

There have been many attempts to fully understand the mechanism of cancer behavior. Yet, how cancers develop andmetastasize still remain elusive. Emerging concepts of cancer biology in recent years have focused on the communicationof cancer with its microenvironment, since cancer cannot grow and live alone. Cancer needs to communicate with othercells for survival, and thus they secrete various messengers, including exosomes that contain many proteins, miRNAs,mRNAs, etc., for construction of the tumor microenvironment. Moreover, these intercellular communications betweencancer and its microenvironment, including stromal cells or distant cells, can promote tumor growth, metastasis, andescape from immune surveillance. In this review, we summarized the role of proteins in the exosome as communicatorsbetween cancer and its microenvironment. Consequently, we present cancer specific exosome proteins and their uniqueroles in the interaction between cancer and its microenvironment. Clinically, these exosomes might provide usefulbiomarkers for cancer diagnosis and therapeutic tools for cancer treatment.

BackgroundCell release diverse types of extracellular vesicles;apoptotic bodies whose sizes are 50 to 5,000nm withtheir irregular lipid bilayers, as well as microvesicleswhose size 50 to 1,000nm is smaller than apoptotic bod-ies but also has an irregular shape. Exosomes are 30-100nm in diameter and contain DNA, miRNA, mRNA,lncRNA, proteins, etc. within their lipid bilayer mem-brane [1–5] (Fig. 1). Apoptotic bodies and microvesiclesare originated from cell membrane surface. Exosomesare smallest extracellular vesicles and originating fromendosomes [6]. Exosomes are secreted by various celltypes and conditions [7]. After being released from thedonor cells the, exosomes travels through the blood andother body fluids. While traveling through the body,exosomes enter the recipient cells through membranefusion and induce transcriptional and, even more abun-dantly, translational changes [8–10]. Tumor cells how-ever secrete more exosomes than normal cells and these

cancer-derived exosomes are involved in tumorigenesis,metastasis and forming the tumor microenvironment[11]. Recently, many researches have revealed that theexosome is a mediator of cell to cell communication andcan be a good candidate for a liquid biopsy biomarker[12–16]. There have been analyses of breast cancer-derived exosomal proteins by liquid chromatography-mass spectrometry (LC-ms/ms), which revealed that theexosome contains a variety of proteins, for example,enzymes, membrane proteins, heat shock proteins, andeven transcription factors. This review discusses cancer-derived exosomal proteins and their roles in the inter-action with tumor microenvironment.

Exosome isolation and protein digestion forproteomicsAfter many research studies proved that exosomes playa role in cell to cell communication through proteins,the interest in exosomes continued growing. However,the method of exosome isolation and analysis is still de-bated [8]. High yield and purity can not only enhancequality but also help us to understand the exosome’s rolein specific conditions. Here, we will discuss exosome

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

* Correspondence: jeycho@snu.ac.kr1Department of Biochemistry, BK21 Plus and Research Institute for VeterinaryScience, School of Veterinary Medicine, Seoul National University, Seoul,South Korea2Department of Biochemistry, College of Veterinary Medicine, Seoul NationalUniversity, 1 Gwanak-ro, Gwanak-gu, Seoul 08826, Korea

Kim et al. Proteome Science (2019) 17:5 https://doi.org/10.1186/s12953-019-0154-z

isolation methods and digestion methods of exosomalproteins from plasma/serum and cells.Various exosome isolation methods have been devel-

oped [8, 17, 18]. Many of these methods can be catego-rized into three main categories according to thecharacteristics of the exosome; density, size, and immu-noaffinity. First, sorting exosome by density is the mostcommon method and utilizes differential centrifugationby varying the g force. Shortly, this is started with centri-fuging at 300-500g to remove cells, accelerating thespeed to 2,000-20,000g to remove cellular debris, and fi-nally speeding up to 100,000-200,000g for the exosomeisolation. Using this method, researchers can get exo-somes in the pellet. However, isolation takes a long timeand requires a lot of input. The biggest drawback is rela-tively low efficiency and poor recovery. Recently, com-mercial precipitation reagents have been developed.Using a precipitate for exosome isolation has a higheryield than using an ultracentrifuge, but lower qualitysince the precipitate can lead to the precipitation of pro-teins. Second, using the smaller than 200nm size charac-teristic of the exosome allows it to be separated byfiltration and size exclusion chromatography. Filtrationand size exclusion chromatography can filter out the cellmembrane, sub-cellular fraction and anything that has abigger size than the exosome. To increase efficiency andpurity, many researchers use a combined method, suchas filtration and ultracentrifuge, or filtration and precipi-tate reagents. Muller et al suggested that this combinedmethod is better than using only one method [19].Lastly, the immunoaffinity for isolation method usesantibodies to capture exosomal proteins. The common

proteins isolated by immunoaffinity are tetraspaninssuch as CD9, CD63, and CD81.After isolation of the exosome, we must lyse the lipid

bilayer membrane and digest proteins to peptides formass spectrometry (MS) analysis. Here, we summarized3 protein digestion methods; In-gel digestion, In-sol di-gestion, and Filter Aided Sample Preparation (FASP)(Fig. 2) [20]. First, in In-gel digestion, lysed exosomalproteins are resolved on a polyacrylamide gel and visual-ized using Coomassie brilliant blue or other stainingreagents. The gel is then sliced to a 1mm size anddestained by ammonium bicarbonate. The next steps arereduction, alkylation, and digestion. Peptides go throughthe process of enrichment and cleanup. Then, dried pep-tides are resuspended and injected into LC-ms/ms [21].Second, for In-sol digestion, lysed exosomes are kept inan aqueous state. Exosomal proteins sequentiallyundergo reduction, alkylation, and digestion in the aque-ous state. Like the in-gel method, peptides are thenenriched and cleaned up before being injected into LC-ms/ms. Lastly, in the FASP method, all of the above-mentioned processes are processed on Microcon 30kcentrifugal ultrafiltration units. Lysed exosomes areloaded onto the filter and discard the elute after centri-fugation. Reduction, alkylation and digestion are allprocessed on the filter. Overall, each method has its ad-vantages and disadvantages. Here, we summarized meth-odological properties in Table 1 [21, 22].Cho (2015) et al., suggested that the biggest issues in

exosome research arise from the exosome isolationmethod. Since the proper isolation method for exosomestudy remains debated however, we summarized exosome

Fig. 1 Schematic description of the extracellular vesicles, Exosomes are smallest extracellular vesicles (30-100nm) secreted from endosomes.Microvesicles are small vesicles (50-1,000nm), and apoptotic bodies are largest extracellular vesicles, both are originated from cell membrane

Kim et al. Proteome Science (2019) 17:5 Page 2 of 14

isolation and exosomal protein digestion methods fromthe studies for exome proteome analyses in Table 2.The exosomes secreted from the cells and biological

fluids are most often separated by a combined method.The most commonly used method is the fusion of ultra-centrifugation and filtration. Exosomes are usuallydigested by the In-Gel , In- sol and FASP methods. Be-fore the FASP method arose [61], the most used methodwas the In-Gel method. But the FASP method is knownto have both the In-Gel and In-Sol methods’ advantages,thus recently manystudies used the FASP method for di-gestion regardless of where exosomes came from.

Cancer-derived exosomal ProteinsBreast cancerBreast cancer is the deadliest cancer in women. One ineight women are diagnosed with breast cancer in theirlifetime [62] and breast cancer accounts for 30% ofnewly diagnosed cancers in women [63]. For the last 10years breast cancers’ death rates and incidence rates inThe United States have risen each year. The exosomehas been revealed as a potential liquid biopsy biomarkerand numerous studies using liquid chromatography-mass spectrometry (LC-ms/ms) have revealed thatcancer derived exosomes contains various proteins,

Fig. 2 Summarization of exosomal protein digestion methods. Exosomes from cell supernatant and body fluid are digested by (1) In-geldigestion (2) In-sol digestion and (3) FASP methods

Table 1 Advantages and Disadvantages of proteomic digestion techniques

Digestion Method Advantages Disadvantages

In-gel Digestion Reproducible, Cost effective, Removal of mass spectrometryincompatible detergents (SDS, Triton etc.) and contaminants,Wide cover range

Time consuming, Inacceptable for extremely acidic or basicand high or low molecular weight proteins and membraneproteins

In-sol Digestion Require less time Inacceptable for low resolubilization proteins

FASP Acceptable for membrane proteins, Removal of massspectrometry incompatible detergents (SDS, Triton etc.)

Loss of proteins , Bad repoducibility, Require large amount ofprotein sample (> 50ug)

Kim et al. Proteome Science (2019) 17:5 Page 3 of 14

including enzymes, membrane proteins, heat shock pro-teins, and even transcription factors.There are many studies on different types of breast

cancer exosomes; the cell line derived exosomes de-scribed, or exosomes derived from breast cancer patientbiological fluids. An early exosome proteome study iden-tified that exosomes derived from breast cancer cell linesMCF-7 and MDA-MB-231 have 59 and 88 proteins, re-spectively [64]. The MDA-MB-231 derived exosomecontained more enzymatic proteins than the MCF7 de-rived exosome. The number of common proteins be-tween the two cell lines are 27. These includecytoskeleton proteins such as β-actin, tubulin-β, andintegrins; membrane proteins like BASP1; enzymes in-cluding enolase α and PRDX1; ribosomal proteins likeRS27A; heat shock proteins including HSP90A, HSP90B,HSP7C; and epigenetic modification related proteinssuch as Histone proteins and 14-3-3 proteins. β-actinand tubulin-β are associated with breast cancer metasta-sis [65, 66]. Overexpression of these proteins in breastcancers show high metastatic potential. It has alreadybeen demonstrated by Hoshino et al that exosomalintegrins α6/β4 and α6/β1 were related to lung metasta-sis and integrin αv/β5 was related to liver metastasis[32]. They also found that exosomal integrins activatethe Src signaling pathway in the recipient cells, whichinduces the inflammatory response. There is no studyyet regarding the correlation of BASP1 and breast can-cer. But there is a study demonstrating that BASP1 over-expression promotes cervical cancer cell progression andcan be a prognostic marker [67]. Enolase α is the glyco-lytic enzyme that catalyzes fructose-1,6-biphosphate toglyceraldehyde 3-phosphate and dihydroxyacetone phos-phate. Research has revealed that an increased level of

enolase α is related to breast cancer metastasis and drugresistance [68, 69]. PRDX1 is an antioxidant enzyme, butits role in breast cancer is controversial. It is, however,clearly overexpressed in breast cancer tissue relative tonormal tissue [70]. Recently, Bajor et al demonstratedthat PRDX1 is involved in reducing exogenous oxidativestress and induces cell growth in breast cancer [71]. TheH2B1 (Histone H2B type 1-C/3/F/G/I) proteins are re-lated to epigenetic regulation. Exosomal histone proteinshave been detected in cancers and other diseases, andeven in normal conditions [72, 73]. However, there arequantitative differences between those detected in cancerversus normal conditions. The role of exosomal histoneproteins in recipient cells is currently controversial, butexosomes also contain 14-3-3 protein, which has beenshown to bind with histone proteins [74, 75]. So, H2B1can potentially induce epigenetic changes in recipientcells by binding with exosomal 14-3-3 proteins.The most abundant MCF-7 derived exosomal proteins

are the structural proteins such as fibronectin, annexinA1, vimentin, actin α, etc., and heat shock proteins.Fibronectin is also known to induce tumor progressionand metastasis. The amount of fibronectin is muchhigher in the exosome of breast cancer patients’ plasmathan normal plasma exosome [76]. A study revealed thatfibronectin secreted by myeloma cell is attached to therecipient cell membrane and turns on p38 and pERKsignaling [77]. Activated p38 and pERK signaling inducesmyeloma cell progression by activating DKK1 andMMP-9. The second largest presence after fibronectin isannexin A1, which is attached to the phospholipid mem-brane. Annexin A1 inhibits phospholipase A2 andinduces anti-inflammatory activity [78]. Similarly, it issuggested that annexin A1 induces metastasis, macro-phage polarization, and poor prognosis [79]. This pro-vides support for Okano et al’s claim that increase in theamount of annexin A1 results in cell invasion which pro-gresses into metastasis [80]. 5’-NTD (5’- nucleosidase,CD73) is the next dominant protein in the exosome ofMCF7 cells. 5’-NTD is the enzyme that catalyzes thecarbon 5’-nucleoside phosphorolytic cleavage and thus isessential for recycling adenosine and cell growth [81]. Itis also overexpressed in many breast cancers. When 5’-NTD is overexpressed in breast cancer cells, it acceler-ated adhesion, migration and invasion of cancer cells[81–84]. So, it can be a clue for how MCF7 changes itmicroenvironment. That is cancer cells secretes a metas-tasis accelerator via the exosome. The 5’-NTD is alsorelated to the immune response [85]. Exosomal 5’-NTDproduces adenosine and indirectly modulates regulatoryT cell (Treg) mediated immunity. Immune modulationfor Treg by 5’-NTD in cancer microenvironment alsohelps cancer cells for their growth and metastasis.Among the 59 proteins identified in the exosome

Table 2. Summarization of used techniques for cancer-derivedexosomes isolation and exosomal protein digestion methods

Origin ofExosome

Isolation Method DigestionMethod

Reference

Cell Ultracentrifuge In-Gel Digestion [23–30]

In-Sol Digestion [26, 31, 32]

Precipitationreagent

In-Gel Digestion [33–37]

FASP [38, 39]

Combined method In-Gel Digestion [28, 35, 36, 40–51]

In-Sol Digestion [52–54]

FASP [51, 55–59]

Plasma/Serum

Ultracentrifuge In-Sol Digestion [60]

FASP [56]

Combined method In-Gel Digestion [43]

In-Sol Digestion [44, 52]

FASP [51, 57, 58]

Kim et al. Proteome Science (2019) 17:5 Page 4 of 14

secreted by MCF7, 30 proteins have been known toparticipate in breast cancer growth, metastasis, andchemoresistance.The most abundant MDA-MD-231 derived exoso-

mal protein is β-actin, which is frequently used as ahousekeeping gene in the exosome. There are alsoother structural proteins in the exosome such astubulin-β and keratins. In general, these cytoskeletalproteins of β-actin, tubulin-β and keratin are all asso-ciated with breast cancer metastasis. Fourteen otherexosomal proteins are also known to be involved inthe metastasis of breast cancer [65, 66, 86]. Tubulin-βis also known to induce chemotherapy resistance [87].Palazzolo et al identified 32 proteins that are moreabundant in the MDA-MB-231 exosome than MCF7cells [88]. Of these 32 proteins, 5 overlap with exoso-mal proteins that Kruger et al identified. These 5 pro-teins, 14-3-3 protein epsilon, β-actin, annexin A1/5,heat shock protein 71 and galectin 3 binding proteincan be potential biomarker candidates for breastcancer-derived exosome. In addition, a study also sug-gested del-1 as an early stage breast cancer exosomebiomarker [89].Klinke et al identified 27 and 28 proteins from other

breast cancer cell lines, BT-474 and SKBR-3, respectivelyby secretome profiling through LC-ms/ms [90]. Somecommon proteins with MCF7 and MDA-MB-231 -de-rived exosomal proteins emerged, for example, β-actin,heat shock proteins, aldolase α, enolase α, 14-3-3 pro-teins, etc. BT-474 and SKBR-3 are HER2 positive celllines, and secreted exosomes highly enriched with pro-teins involved in antigen presenting (HSPA5, CALR,PSME1,2, PSMA 3,6, PSMB 2,4, and HLA-C) and glyco-lytic metabolism (G6PD, TP1, and PGAM1). These pro-teins could lead to cancer immune-surveillance andmalfunctioned energy synthesis in breast cancer micro-environment [91–93]. In addition, BT-474-derived exo-somal proteins have a strong relation with neutrophilGO terms; DDX3X, VCP, HSP90AA1, ILF2, HSPA8,PNP, MME, MME2, PAB37, SERPINB6, GDI2, ALDOA,PGAM1, and GPI, which are related with neutrophil de-granulation, mediated immunity and neutrophil activa-tion. Immune suppression by cancer exosomes and itsrelation to neutrophils have already been studied [94]. Ina breast cancer-bearing mice model, neutrophils wereactivated and exosome levels in blood were much higherthan the normal control group. It is also suggested thatexosomes derived from tumors interact with neutrophilsand induce cancer-associated thrombosis. All these evi-dences strongly suggest the importance of cancer-derived exosome in the immune modulation.In addition, exosomes are thought to help increase

breast cancer tumorigenesis. This is due to the specifi-city of the proteins found in the exosomes. Firstly, the

exosomes isolated from the serum of breast cancer pa-tients had high amounts of survivin [95], a protein thatcontrols anti-apoptosis of the surrounding cells, andexosomes isolated from the cell line had large amountsof MTA1 [96], a protein that promotes proliferation, .Secondly, there is a large amount of drug and chemore-sistance proteins in the exosome. GSTP1, TGF-β1,TPRC5, and UCH-L1 are examples [97–100]. Finally,proteins that induce metastasis present highly in exo-somes. A typical example is nephronectin, which hasbeen reported to be high in the serum of patients withmetastases [101]. It is also notable that analysis of breastcancer cells and their metastasized cancer cell derivedexosomes revealed that integrin α6/β4, caveolin-1, peri-ostin and myoferlin are more enriched in metastasizedcancer cells than primary breast cancer cells [31, 32, 36,102], although their roles need to be further investigated.These proteins might serve as biomarker candidates forbreast cancer.

Lung CancerLung cancer is the most common cause of cancer re-lated death in both sexes and also shows the highest in-cidence rate among cancer in the United States [63].Non-small cell lung cancer (NSCLC) accounts for thelargest proportion of lung cancer patients. This is furthercategorized into adenocarcinoma, squamous cell carcin-oma, and large cell carcinoma. Lung cancer is foundlater than any other cancer because it has no symptomsthat can be discerned through self-awareness. Thus, re-gardless of the many developed treatments for lung can-cer, after diagnosis it is often too late to treat. This leadsto a poor 5-year survival rate and motivates the searchfor scanning biomarkers. Here, we classify andsummarize the roles of the NSCLC-derived exosomalproteins.Clark et al analyzed two of the NSCLC cell lines, A549

and HCC827 [51]. They normalized the LC-ms/ms datawith normal lung cell line HBE3. The number of pro-teins that expressed twice more in A549 than normalHBE3 is 58. Mucin 5 AC and B proteins are highlyenriched in A549 derived exosome. Mucin 5 AC and Bare known to only be expressed in lung adenocarcinoma.Overexpression of these proteins leads to lung cancer re-lapse and metastasis [103, 104]. Furthermore, there areAnnexin proteins, ADAM10, EGFR, integrin, JAK, andmetabolism related enzymes found in the A549 derivedexosome. Of the 58 proteins, 12 (20%) are correlatedwith neutrophil degranulation and neutrophil-mediatedimmunity (JUP, C3, VCP, CD44, etc.). The HCC827 exo-some has 93 more proteins than the HBE3 derived exo-some. The most abundant protein is desmoglein-2. Ithas been reported that desmoglein-2 is overexpressed inNSCLC tissues and induce NSCLC growth by regulating

Kim et al. Proteome Science (2019) 17:5 Page 5 of 14

p27 and CDK2 [105]. The next most enriched protein isEGFR. Given that EGFR is an oncogene in lung cancer,this can be a powerful clue to explain the exosome’sextreme tumorigenicity role in the neighboring micro-environment [106]. HCC827 derived exosomes also con-tain several kinds of other proteins; Integrin, Annexinproteins, guanine nucleotide-binding proteins (GPCR),and 14-3-3 proteins. Like in the A549 derived exosomalproteins, there are also neutrophil-related proteinsenriched in the HCC287 derived exosome as well(22.5%).The exosomes secreted by lung cancer, like the exo-

somes secreted by breast cancer, are also involved intumorigenesis. Most cancers are characterized by metas-tasis only to certain organs, called organotropic metasta-sis [107]. Recent studies suggest that exosomes are alsoinvolved in organotropic metastasis [32]. Integrin plays avery important role. Hoshino et al revealed that treat-ment of exosomes isolated from lung cancer cells redi-rects lung cancer cells to metastasize to bone [32].Exosomal integrins α6β4 and α6β1 are involved in lungcancer organotropic metastasis. As such, proteins in exo-somes can induce metastasis by reprogramming cells.Exosomal Leucine-Rich-Alpha2-Glycoprotein 1 (LRG1)secreted from lung cancer cells induces angiogenesisthrough TGF-beta signaling in recipient cells [108]. Thisexosomal protein is also found in exosomes isolatedfrom urine in patients with lung cancer [109], whichmay serve as a good prognostic marker. Anotherexample is T-cell immunoglobulin- mucin-domain-containing molecule 3 (Tim-3) and its ligand Galectin-9(Gal-9). Tim3 and Gal-9 exhibit anti-tumor immune re-sponses, by blocking Th1 type immune responses [110].Both proteins were found to be higher in the plasma de-rived exosomes of lung squamous cell carcinoma pa-tients than in lung adenocarcinoma patients [110]. It isnot yet known why these two proteins are contained inthe exosome. Yet Tim-3 and Gal-9, can be used as prog-nostic and diagnostic markers of lung cancers.NY-SEO-1, EGFR, PLAP, and EpCam were high in

exosomes isolated from the plasma of lung cancer pa-tients [111], and Vykoukal et al also revealed that SGRN,TPM3, THBS1, and HUWE1 levels in plasma-derivedexosomes in lung cancer patients is higher than controlgroup [112]. EGFR is a protein that is highly related tolung cancer, and is abundant in exosomes isolated fromlung cancer cells, lung biopsies and plasma, thus makingit the most powerful biomarker from the revealed candi-dates [111, 113, 114]. Exosomes derived from NCI-H838, another NSCLC cell line, contain more MUC1 asrevealed in patients’ plasma exosomes. Other report alsorevealed that MUC1 in NSCLC patient plasma exosomeas much higher than that in the normal control groupplasma exosome [115].

Other cancersAmong all women and men diagnosed with cancer eachyear, there is a high proportion of people diagnosed withcolon cancer [63]. Colon cancer cell derived exosomalproteins are identified by Choi et al [42] and Mathivananet el [33]. The common proteins of both studies are re-lated with cancer progression and metastasis. Choi et alalso suggested that the identified proteins have relationwith immune modulation. Furthermore, human coloncancer ascites derived exosomes have similar tumorigen-esis potential with proteins related to cancer progres-sion, immune modulation, and metastasis [116]. Notonly ascites, but also serum exosome can be a gooddiagnostic marker. Annexin proteins, and tspan 1 fromthe serum exosome are suggested colon cancer diagnos-tic markers [35, 117]. However, most cancer derivedexosomes, as mentioned in breast and lung cancer de-rived exosomes as above, contain a lot more annexinand tetraspanin proteins than normal control derivedexosome. So, these two proteins rather are pan-cancerexosome proteins.Pancreatic cancer incidence and death rate have been

increasing. Moreover, the 5-year relative survival rates ofpancreatic cancer is only 9%, whereas other cancers suchas prostate is 98% and melanoma is 92% [63]. Pancreaticcancer cell derived exosome that induce metastasis [118,119] and chemoresistance [27] have been revealed. Sev-eral proteins, glypican-1, CD44, Tspan8, EpCam, METand CD104, have been suggested as pancreatic cancerexosome-derived biomarkers [31, 120]..Renal cancer incidence rate is 3 to 5 % for both males

and females, but there is no accurate biomarker for renalcancer. Raimondo et al identified renal cancer patients’urinary exosomes [121]. They suggested 10 proteins forrenal cancer exsome biomarker, 5 of which are abundantin renal cancer patients; matrix metalloproteinase 9(MMP-9), ceruloplasmin (CP), podocalyxin (PODXL),dickkopf related protein 4 (DKK4) and carbonic anhydraseIX (CAIX). Oppositely, aquaporin-1 (AQP1), extracellularMatrix metalloproteinase Inducer (EMMPRIN), neprilysin(CD10), dipeptidase 1 and syntenin-1 are abundant in theexosome of healthy control. They claimed that these 10proteins have great potential for early stage diagnosis ofrenal cancer with clinical value. A summary of selectedexosomal proteins is given in Table 3.

The role of exosome proteins in tumormicroenvironment: Friend or Foe ?Tumor microenvironmentMany approaches to conquer cancer are ongoing all overthe world. Nevertheless, incidence and death rates ofcancer are on the rise every year [63]. The main cause ofthe increasing incidence rate and mortality is not onlyprimary tumors, but also distant tumors [122]. Many

Kim et al. Proteome Science (2019) 17:5 Page 6 of 14

Table 3 Description of the selected exosomal proteins incancer

Exosomal Protein Description

Breast Cancer

β-actin Breast cancer metastasis [65]

Tubulin-β Breast cancer metastasis,chemotheray resistance [66, 87]

Integrin α6/β4, α6/β1 Lung metastasis [32]

Integrinαv/β5 Liver metastasis [32]

BASP1 Overexpression leads to ovariancancer cell progression [67]

Enolase A Breast cancer metastasis and drugresistance [68, 69]

PRDX1 Induce cell growth in breast cancerand overexpressed in breastcancer [70]

14-3-3 protein Bind to histone protiens and induceepigenetic changes [75]

Fibronectin Tumor progression andmetastasis [76]

Annexin A1 Induce tumor metastasis andmacrophage polarization [78–80]

5’-NTD Overexpressed in breast cancer cellsand induce metastasis [81–85]

Survivin Overexpressed in breast cancerserum derived exoxome, Antiapoptosis [95]

MTA1 Promote proliferation [96]

GSTP1 Drug and chemotherapyresistance [97]

TGF-β1 Drug resistance [98]

TPRC5 Chemotherapy resistance [99]

UCH-L1 Chemotherapy resistance [100]

Nephronectin Induce tumor mestasis [101]

Caveolin-1 Enriched in metastasized cancercell [36]

Periostin Enriched in metastasized cancercell [102]

Myoferlin Enriched in metastasized cancercell [31]

Lung Cancer

Mucin 5AC, B Lung cancer relapse andmetastasis [103, 104]

Desmoglein-2 overspressed in non small celllung cancer and induce cellgrowth [105]

EGFR Oncogene in lung cancer[106, 111, 113, 114]

LRG1 Induce angiogenesis [108]

Tim-3 Induce anti-tumor immuneresponse [110]

NY-SEO-1 Overexpressed in lung cancer [111]

Table 3 Description of the selected exosomal proteins incancer (Continued)Exosomal Protein Description

PLAP Overexpressed in lung cancer [111]

EpCam Overexpressed in lung cancer [111]

SGRN Overexpressed in lung cancer [112]

TPM3 Overexpressed in lung cancer [112]

THBS1 Overexpressed in lung cancer [112]

HUWE1 Overexpressed in lung cancer [112]

MUC 1 Overexpressed in lung cancer [112]

Colon Cancer

Annexin family Colon cancer progression andmetastasis [33, 42]

Tetraspanin 1 Colon cancer progression andmetastasis [33, 42]

Pancreatic Cancer

Glypican-1 Abundant in pancreatic cancerexosome [31, 120]

CD44 Abundant in pancreatic cancerexosome [31, 120]

Tspan 8 Abundant in pancreatic cancerexosome [31, 120]

EpCam Abundant in pancreatic cancerexosome [31, 120]

MET Abundant in pancreatic cancerexosome [31, 120]

CD104 Abundant in pancreatic cancerexosome [31, 120]

Renal Cancer

MMP-9 Abundant in renal cancerexosome [121]

CP Abundant in renal cancerexosome [121]

PODXL Abundant in renal cancerexosome [121]

DKK4 Abundant in renal cancerexosome [121]

CAIX Abundant in renal cancerexosome [121]

AQP1 Abundant in renal cancerexosome [121]

EMMPRIN Abundant in renal cancerexosome [121]

CD10 Abundant in renal cancerexosome [121]

Dipeptidase 1 Abundant in renal cancerexosome [121]

Syntenin-1 Abundant in renal cancerexosome [121]

Kim et al. Proteome Science (2019) 17:5 Page 7 of 14

researchers have been trying to understand the mechan-ism of metastasis to find a cure the cancer [123–125]. Arising concept for the metastasis mechanism is that thetumor is collaborating with the tumor microenviron-ment through exosomes.The tumor microenvironment consists of immune

cells, fibroblasts, the extracellular matrix, basementmembrane, endothelial cells, and cancer cells [126–129].Several roles of the tumor microenvironment have beensuggested [126]. Component cells of the tumor micro-environment have roles in tumor initiation, progression,and metastasis. Many studies have revealed that thecomponents of the tumor microenvironment communi-cate via exosomes [1, 130–134]. Here, we summarizedpotential roles of the exosome between cancer cells andtumor microenvironment cells, and the effect on tumori-genesis (Fig. 3).

Cancer associated fibroblast (CAF)A predominant stromal cell component of the tumormicroenvironment is activated by a fibroblast, termedcancer-associated fibroblast (CAF) [135]. Several studieshave revealed that CAFs are highly involved in tumorprogression [136–138]. Mammary carcinoma-derivedexosomes induce mammary fibroblast motility by trans-ferring AHNAK [139]. As such, the exosome secreted bycancer cells can affect fibroblasts, but here we also dis-cuss how fibroblast-derived exosome can affect cancercells. Many researchers have suggested that CAFs have arelation to cancer proliferation, chemoresistance, andmetastasis. Takasugi et al demonstrated that senescent

fibroblast-derived exosome can induce MCF7 cell prolif-eration by transferring EphA2 [140]. In pancreatic can-cer, it is reported that chemotherapy stimulated CAFs torelease more exosomes, which in turn promoted recipi-ent cancer epithelial cells’ proliferation and drug resist-ance [141]. Exosomes secreted by CAFs also induceepithelial-mesenchymal transition (EMT), migration, andinvasion, resulting in metastasis and cell growth of blad-der cancer by activating IL-6 signaling [142]. TGFβ1 isenriched in ovarian CAFs and affects ovarian cancercells into EMT by SMAD signaling activation [143]. Inlung cancer, CAF-derived exosome also enhance metas-tasis by activation of the IL-6/STAT3 signaling pathway[144]. Furthermore, exosomes secreted by CAFs canaffect chemotherapy resistance [145]. It is suggestedCAFs-derived exosome in colorectal cancer stem cellscan promote drug resistance and enhance cancer stemcell properties and also growth.

Natural killer (NK) cellNatural killer (NK) cells are large granular cytotoxiclymphocytes that kill the target cancer cells withoutstimuli. There are several mechanisms by which NKcell-derived exosomes kill the recipient cells [146]. Acti-vated NK cell-derived exosomes are cytotoxic becausethey can induce the cell death pathway by perforin(PFN), granzymes (Gzm-A/B) and granulysin (GNLY).Wen et al suggested that activated NK cell exosomes de-liver caspase inducers which lead to cancer death by ac-tivation of the caspase-dependent cell death pathway.Perforin is delivered to form the pores and granzymes

Fig. 3 The function of cancer and other components of tumor microenvironment-derived exosome. Cancer-derived exosomes contain varioustypes of proteins for immune suppression, cancer progression and metastasis

Kim et al. Proteome Science (2019) 17:5 Page 8 of 14

enter into the recipient cells. Granzymes induce caspase-dependent and -independent cell death. Cancer cellsincubated with activated NK cells showed an increasedexpression of activated caspase-3, -7, and -9 [147]. Pros-tate cancer-derived exosomes downregulate NKG2Dexpression on NK cells and CD8+ T-cells. This leads todownregulation of NKG2D-mediated cytotoxic responsein prostate cancer patients via immune escape [148].Berchem et al showed that hypoxic tumor-derived exo-somes transfer TGF-β1 to NK cells, which leads to thedown regulation of NK cell’s surface expression ofNKG2D receptor [149]. Decreasing NKG2D inhibits NKcell function. Renal cancer cell-derived exosomes areenriched with TGF-β1 and activate the TGF-β1/SMADpathway in NK cells to facilitate immune escape [150].Another mechanism of NK cell immune response escapeis related to p75NTR. NK cells in tumor microenviron-ment have high expression of p75NTR and exosomes se-creted by lung cancer contain proNGF and sortilin,which bind to p75NTR and induce NK cell apoptosis[151]. Interestingly, exosomes released from cancer cellsdo not always inhibit the activity of NK cells. Wang et alrevealed that ovarian cancer cell-derived exosomes en-hance the cytotoxicity effect of NK cells [152]. Exosomessecreted by ovarian cancer cell contain phosphorylatedIRF-3 that promotes NK cell cytotoxicity by inducinginterferon gene expression in NK cells.

T-cellCancer-derived exosomes are known as immune sup-pressors because they can inactivate effector T cells andinduce T cell apoptosis. There were a lot of researchconducted on cancer-derived exosomes and T cell inter-action. Cancer-derived exosomes could induce T cellsuppression by delivering Fas ligand (FasL), PD-L1,TGF-β, adenosine and galectin-9. Abusamra et al dem-onstrated that prostate cancer cell-derived exosomeshave FasL that induces T cell apoptosis upon delivery bycaspase activation [153]. Colorectal cancer-derived exo-somes have shown the same effect on T cell apoptosis[154]. The exosome isolated from head and neck cancerpatients’ serum also induces T cell apoptosis [155].Tumor cells expressing PD-L1 in their membrane canescape form the immune response. PD-L1 is also de-tected in cancer-derived exosomes. Prostate and melan-oma cancer cell-derived exosomal PD-L1 binds toeffector T cell’s membrane PD-1 receptor, therebyimpairing their growth [156]. Melanoma patients’ circu-lating exosomes also have PD-L1, which also inducesimmune surveillance.Exosomal TGF-β 1 is a well-known immune surveil-

lance factor [157]. Hypoxia conditioned BT-474 andMDA-MB-231 secreted more exosomes than normoxia

condition. These exosomes have an increase in TGF-βwhich inhibits T cell proliferation. Colorectal cancer-derived exosomes were enriched with TGF-β1, which in-duces alteration of T cell phenotype to T regulatory cellsby activating TGF-β/Smad signaling and inactivatingSAPK signaling [158]. Prostate cancer cell line-derivedexosomes also contribute to immune evasion by trans-ferring exosomal TGP-β1 [159].Adenosine is also a key factor in a known pathway that

induces T cell suppression. Tumor exosomes have beenknown to contain CD39 and CD73 on the membrane sur-face. Exosome-mediated transfer of CD39 and CD73 leadsto hydrolysis of ATP to adenosine. Accumulated adeno-sines participate in T cell inactivation by binding with theirreceptors (A1, A2A, A2B, and A3) [160]. Adenosine bindstheir receptors in Treg cells and triggers the cyclic AMP(cAMP) and protein kinase A (PKA) signal [161]. This sig-nal can regulate either survival or apoptosis of the T cell,depending on the signal strength and duration [162].Galectin also has a role in tumor-derived exosome

induced immune escape. Galectin-1 is a type of β-galactosidase protein expressed in immune cells. Recently,it has been shown that tumors secreting galectin-1 on ahigh level affect immune cells by binding to N-acetyllactosamine on the T cell membrane. Interaction ofgalectin-1 and the galectin ligand induces immune escapethrough T cell apoptosis [163]. It is also demonstrated en-richment of galectin in head and neck cancer-derived exo-some induced suppression of CD8+ T cell [164].

MacrophageMacrophages belong to the innate immune system. Macro-phages are divided into two types by Th1 and Th2polarization, which are called M1 and M2, and have thecharacteristics of pro-inflammatory and anti-inflammatory,respectively [165]. Tumor-associated macrophages (TAMs)consist of M2 characteristic macrophages and promoteangiogenesis, invasion, and metastasis [166]. M2 macro-phages secrete tumor metastasis supporting cytokines suchas CCL2, MIP2, IL-8, and IL-Rα and attenuating antitumorcytokines such as TIMP-1, IFN-γ, IL-1Rα, IL-13, and IL-16[167, 168]. Chen et al described how colorectal cancer-derived exosomes induce M2 macrophages by cytoskeletonrearrangement [169]. Gastric cancer-derived exosomes alsohave effect to M2 macrophage [170].On the other hand, it has been also reported that gas-

tric cancer-derived exosomes activate the NF-kB path-way in recipient macrophages, leading to up-regulationof pro-inflammatory factors [171] such as IL-6 andTNF-α which promote gastric cancer progression [172].A similar result was also reported that breast cancer cellline-derived exosome stimulates the NF-kB pathway inmacrophages, which leads to the secretion of pro-inflammatory factors such as IL-6, TNF-α, GCSF, and

Kim et al. Proteome Science (2019) 17:5 Page 9 of 14

CCL2 [173]. Breast cancer exosomal protein HSP72 andRNAs are involved in stimulating the NK-kB pathway. Itwas also demonstrated that breast cancer-derived exo-somes promote macrophage polarization and inducelymph node metastasis [174]. It has been also shownthat breast cancer-derived exosomes are enriched withgp130, which induces gp130/STATS signaling in macro-phage and leads to the secretion of IL-6 for macrophagepolarization [175]. From this point of view, tumor-derived exosomes are assumed to play both anti-inflammatory and pro-inflammatory roles by stimulatingM2 and M1 macrophages, respectively.TAM-derived exosomes also participate in tumorigen-

esis. For example, TAM-derived exosomes enhancetumor invasion by delivering wnt5a in macrophages tobreast cancer cells. Delivered wnt5a enhances tumor in-vasion by leading to the activation of β-catenin-independent Wnt signaling [176].

ConclusionSince exosomes are known as cellular communicators,there are many approaches for isolation and exosomalcontent analysis. Sadly, the big hurdles of exosome re-search still remain. The establishment of isolation anddigestion standards remain essential. In this review, webriefly summarized the current methods of exosome iso-lation and its protein digestion. For cell supernatant exo-some analysis, exosomes are isolated by combinedmethods and proteins are digested mostly by In-Gel di-gestion and FASP. Body fluid exosomes are isolated bythe same methods, but proteins are digested by FASPfor mass spectrometry.Cancer-derived exosomes contain various proteins.

Exosomal proteins from cancer cells affect the tumormicroenvironment in their favor through suppressivemodulation of immune cells including NK cells, Tcells, and Macrophages and immune surveillance.Cancer stem cell progression and chemotherapy re-sistance are acquired by modulating cancer associatedfibroblasts. Together, cancer-derived exosomes andtumor microenvironment cell-derived exosomes alterthe cancers to be more aggressive and be able tometastasize. In the process, these tumor-derived exo-somes are enriched and can be detected in biologicalfluids. Recent discoveries in exosome fields will alsoalter cancer management. Indeed, non-invasive diag-nosis and prognosis could become possible via plasmaexosomes. Thus, it is suggested that strategies basedon the blocking of exosomal immune suppressioncould be developed for the treatment of cancer pa-tients. Since exosome fields are expanding, further ef-forts to reveal fundamental mechanisms of exosomecargo selection and biogenesis are necessary to fullyunderstand the roles of proteins in exosomes.

AcknowledgementsWe thank Johannes Josephus Schabort (Department of VeterinaryBiochemistry, Seoul National University, Seoul, Korea) his English grammaticalcorrections of this manuscript.

Authors’ contributionsHJC contributed in the conceptions and approval of this manuscript. HK, DKand JC drafted and wrote the manuscript. All authors read and approved thefinal manuscript.

FundingThis research was supported by the Bio & Medical Technology DevelopmentProgram of the National Research Foundation (NRF) funded by the Ministryof Science and ICT (#2016M3A9B6026771 & #2014M3A9D5A01073598).

Availability of data and materialsNot applicable.

Ethics approval and consent to participateNot applicable.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Received: 12 June 2019 Accepted: 15 October 2019

References1. Maia J, Caja S, Strano Moraes MC, Couto N, Costa-Silva B. Exosome-Based

Cell-Cell Communication in the Tumor Microenvironment. Front Cell DevBiol. 2018;6:18.

2. Seo N, Akiyoshi K, Shiku H. Exosome-mediated regulation of tumorimmunology. Cancer Sci. 2018;109:2998–3004.

3. Tao L, Shi J, Yang X, Yang L, Hua F. The Exosome: a New Player in DiabeticCardiomyopathy. J Cardiovasc Transl Res. 2019;12:62–7.

4. Das CK, Jena BC, Banerjee I, Das S, Parekh A, Bhutia SK, Mandal M. Exosomeas a Novel Shuttle for Delivery of Therapeutics across Biological Barriers. MolPharm. 2019;16:24–40.

5. Wortzel I, Dror S, Kenific CM, Lyden D. Exosome-Mediated Metastasis:Communication from a Distance. Dev Cell. 2019;49:347–60.

6. Chen Y, Li G, Liu ML. Microvesicles as Emerging Biomarkers and TherapeuticTargets in Cardiometabolic Diseases. Genomics Proteomics Bioinformatics.2018;16:50–62.

7. Kourembanas S. Exosomes: vehicles of intercellular signaling, biomarkers,and vectors of cell therapy. Annu Rev Physiol. 2015;77:13–27.

8. Sunkara V, Woo HK, Cho YK. Emerging techniques in the isolation andcharacterization of extracellular vesicles and their roles in cancer diagnosticsand prognostics. Analyst. 2016;141:371–81.

9. Melo SA, Sugimoto H, O'Connell JT, Kato N, Villanueva A, Vidal A, Qiu L,Vitkin E, Perelman LT, Melo CA, et al. Cancer exosomes perform cell-independent microRNA biogenesis and promote tumorigenesis. Cancer Cell.2014;26:707–21.

10. Rana S, Malinowska K, Zoller M. Exosomal tumor microRNA modulatespremetastatic organ cells. Neoplasia. 2013;15:281–95.

11. Henderson MC, Azorsa DO. The genomic and proteomic content of cancercell-derived exosomes. Front Oncol. 2012;2:38.

12. Properzi F, Logozzi M, Fais S. Exosomes: the future of biomarkers inmedicine. Biomark Med. 2013;7:769–78.

13. Alegre E, Zubiri L, Perez-Gracia JL, Gonzalez-Cao M, Soria L, Martin-Algarra S,Gonzalez A. Circulating melanoma exosomes as diagnostic and prognosisbiomarkers. Clin Chim Acta. 2016;454:28–32.

14. Roberson CD, Atay S, Gercel-Taylor C, Taylor DD. Tumor-derived exosomesas mediators of disease and potential diagnostic biomarkers. CancerBiomark. 2010;8:281–91.

15. Taylor DD, Gercel-Taylor C. MicroRNA signatures of tumor-derived exosomesas diagnostic biomarkers of ovarian cancer. Gynecol Oncol. 2008;110:13–21.

16. Tavakolizadeh J, Roshanaei K, Salmaninejad A, Yari R, Nahand JS, SarkariziHK, Mousavi SM, Salarinia R, Rahmati M, Mousavi SF, et al. MicroRNAs and

Kim et al. Proteome Science (2019) 17:5 Page 10 of 14

exosomes in depression: Potential diagnostic biomarkers. J Cell Biochem.2018;119:3783–97.

17. Witwer KW, Buzas EI, Bemis LT, Bora A, Lasser C, Lotvall J, Nolte-'t Hoen EN,Piper MG, Sivaraman S, Skog J, et al. Standardization of sample collection,isolation and analysis methods in extracellular vesicle research. J ExtracellVesicles. 2013;2(1):20360.

18. Yamashita T, Takahashi Y, Nishikawa M, Takakura Y. Effect of exosomeisolation methods on physicochemical properties of exosomes andclearance of exosomes from the blood circulation. Eur J Pharm Biopharm.2016;98:1–8.

19. Muller L, Hong CS, Stolz DB, Watkins SC, Whiteside TL. Isolation ofbiologically-active exosomes from human plasma. J Immunol Methods.2014;411:55–65.

20. Choksawangkarn W, Edwards N, Wang Y, Gutierrez P, Fenselau C.Comparative study of workflows optimized for in-gel, in-solution, and on-filter proteolysis in the analysis of plasma membrane proteins. J ProteomeRes. 2012;11:3030–4.

21. Kim YI, Cho JY. Gel-based proteomics in disease research: Is it still valuable?Biochim Biophys Acta Proteins Proteom. 1867;2019:9–16.

22. Ludwig KR, Schroll MM, Hummon AB. Comparison of In-Solution, FASP, andS-Trap Based Digestion Methods for Bottom-Up Proteomic Studies. JProteome Res. 2018;17:2480–90.

23. Soekmadji C, Riches JD, Russell PJ, Ruelcke JE, McPherson S, Wang C,Hovens CM, Corcoran NM. Australian Prostate Cancer Collaboration B, HillMM, Nelson CC: Modulation of paracrine signaling by CD9 positive smallextracellular vesicles mediates cellular growth of androgen deprivedprostate cancer. Oncotarget. 2017;8:52237–55.

24. Gangoda L, Liem M, Ang CS, Keerthikumar S, Adda CG, Parker BS,Mathivanan S. Proteomic Profiling of Exosomes Secreted by Breast CancerCells with Varying Metastatic Potential. Proteomics. 2017;17(23-24). https://doi.org/10.1002/pmic.201600370.

25. Bisaro B, Mandili G, Poli A, Piolatto A, Papa V, Novelli F, Cenacchi G, Forni M,Zanini C. Proteomic analysis of extracellular vesicles from medullospheresreveals a role for iron in the cancer progression of medulloblastoma. MolCell Ther. 2015;3:8.

26. Garnier D, Magnus N, Meehan B, Kislinger T, Rak J. Qualitative changes inthe proteome of extracellular vesicles accompanying cancer cell transitionto mesenchymal state. Exp Cell Res. 2013;319:2747–57.

27. Fan J, Wei Q, Koay EJ, Liu Y, Ning B, Bernard PW, Zhang N, Han H, Katz MH,Zhao Z, Hu Y. Chemoresistance Transmission via Exosome-Mediated EphA2Transfer in Pancreatic Cancer. Theranostics. 2018;8:5986–94.

28. Ono K, Eguchi T, Sogawa C, Calderwood SK, Futagawa J, Kasai T, Seno M,Okamoto K, Sasaki A, Kozaki KI. HSP-enriched properties of extracellularvesicles involve survival of metastatic oral cancer cells. J Cell Biochem. 2018;119:7350–62.

29. Greening DW, Ji H, Chen M, Robinson BW, Dick IM, Creaney J, Simpson RJ.Secreted primary human malignant mesothelioma exosome signaturereflects oncogenic cargo. Sci Rep. 2016;6:32643.

30. Yi H, Zheng X, Song J, Shen R, Su Y, Lin D. Exosomes mediated pentosephosphate pathway in ovarian cancer metastasis: a proteomics analysis. IntJ Clin Exp Pathol. 2015;8:15719–28.

31. Blomme A, Fahmy K, Peulen O, Costanza B, Fontaine M, Struman I, Baiwir D,de Pauw E, Thiry M, Bellahcene A, et al. Myoferlin is a novel exosomalprotein and functional regulator of cancer-derived exosomes. Oncotarget.2016;7:83669–83.

32. Hoshino A, Costa-Silva B, Shen TL, Rodrigues G, Hashimoto A, Tesic Mark M,Molina H, Kohsaka S, Di Giannatale A, Ceder S, et al. Tumour exosomeintegrins determine organotropic metastasis. Nature. 2015;527:329–35.

33. Mathivanan S, Lim JW, Tauro BJ, Ji H, Moritz RL, Simpson RJ. Proteomicsanalysis of A33 immunoaffinity-purified exosomes released from the humancolon tumor cell line LIM1215 reveals a tissue-specific protein signature. MolCell Proteomics. 2010;9:197–208.

34. Leca J, Martinez S, Lac S, Nigri J, Secq V, Rubis M, Bressy C, Serge A, LavautMN, Dusetti N, et al. Cancer-associated fibroblast-derived annexin A6+extracellular vesicles support pancreatic cancer aggressiveness. J Clin Invest.2016;126:4140–56.

35. Lee CH, Im EJ, Moon PG, Baek MC. Discovery of a diagnostic biomarker forcolon cancer through proteomic profiling of small extracellular vesicles.BMC Cancer. 2018;18:1058.

36. Campos A, Salomon C, Bustos R, Diaz J, Martinez S, Silva V, Reyes C, Diaz-Valdivia N, Varas-Godoy M, Lobos-Gonzalez L, Quest AF. Caveolin-1-

containing extracellular vesicles transport adhesion proteins and promotemalignancy in breast cancer cell lines. Nanomedicine. 2018;13:2597–609.

37. Turay D, Khan S, Diaz Osterman CJ, Curtis MP, Khaira B, Neidigh JW, MirshahidiS, Casiano CA, Wall NR. Proteomic Profiling of Serum-Derived Exosomes fromEthnically Diverse Prostate Cancer Patients. Cancer Invest. 2016;34:1–11.

38. Sun Y, Zheng W, Guo Z, Ju Q, Zhu L, Gao J, Zhou L, Liu F, Xu Y, Zhan Q,et al. A novel TP53 pathway influences the HGS-mediated exosomeformation in colorectal cancer. Sci Rep. 2016;6:28083.

39. Zhang J, Lu S, Zhou Y, Meng K, Chen Z, Cui Y, Shi Y, Wang T, He QY. Motilehepatocellular carcinoma cells preferentially secret sugar metabolismregulatory proteins via exosomes. Proteomics. 2017;17(13-14):1700130.

40. Chaiyawat P, Weeraphan C, Netsirisawan P, Chokchaichamnankit D,Srisomsap C, Svasti J, Champattanachai V. Elevated O-GlcNAcylation ofExtracellular Vesicle Proteins Derived from Metastatic Colorectal CancerCells. Cancer Genomics Proteomics. 2016;13:387–98.

41. Choi DY, You S, Jung JH, Lee JC, Rho JK, Lee KY, Freeman MR, Kim KP, Kim J.Extracellular vesicles shed from gefitinib-resistant nonsmall cell lung cancerregulate the tumor microenvironment. Proteomics. 2014;14:1845–56.

42. Choi DS, Lee JM, Park GW, Lim HW, Bang JY, Kim YK, Kwon KH, Kwon HJ,Kim KP, Gho YS. Proteomic analysis of microvesicles derived from humancolorectal cancer cells. J Proteome Res. 2007;6:4646–55.

43. Lee JE, Moon PG, Cho YE, Kim YB, Kim IS, Park H, Baek MC. Identification ofEDIL3 on extracellular vesicles involved in breast cancer cell invasion. JProteomics. 2016;131:17–28.

44. Griffiths SG, Cormier MT, Clayton A, Doucette AA. Differential ProteomeAnalysis of Extracellular Vesicles from Breast Cancer Cell Lines by ChaperoneAffinity Enrichment. Proteomes. 2017;5(4):25.

45. Klein-Scory S, Tehrani MM, Eilert-Micus C, Adamczyk KA, Wojtalewicz N,Schnolzer M, Hahn SA, Schmiegel W, Schwarte-Waldhoff I. New insights inthe composition of extracellular vesicles from pancreatic cancer cells:implications for biomarkers and functions. Proteome Sci. 2014;12:50.

46. Choi DS, Choi DY, Hong BS, Jang SC, Kim DK, Lee J, Kim YK, Kim KP, Gho YS.Quantitative proteomics of extracellular vesicles derived from humanprimary and metastatic colorectal cancer cells. J Extracell Vesicles. 2012;1.https://doi.org/10.3402/jev.v1i0.18704.

47. Ji H, Greening DW, Barnes TW, Lim JW, Tauro BJ, Rai A, Xu R, Adda C,Mathivanan S, Zhao W, et al. Proteome profiling of exosomes derived fromhuman primary and metastatic colorectal cancer cells reveal differentialexpression of key metastatic factors and signal transduction components.Proteomics. 2013;13:1672–86.

48. Mariscal J, Fernandez-Puente P, Calamia V, Abalo A, Santacana M, Matias-Guiu X, Lopez-Lopez R, Gil-Moreno A, Alonso-Alconada L, Abal M.Proteomic Characterization of Epithelial-Like Extracellular Vesicles inAdvanced Endometrial Cancer. J Proteome Res. 2019;18:1043–53.

49. Suwakulsiri W, Rai A, Xu R, Chen M, Greening DW, Simpson RJ. Proteomicprofiling reveals key cancer progression modulators in shed microvesiclesreleased from isogenic human primary and metastatic colorectal cancer celllines. Biochim Biophys Acta Proteins Proteom. 2018:140171. https://doi.org/10.1016/j.bbapap.2018.11.008.

50. Jerez S, Araya H, Thaler R, Charlesworth MC, Lopez-Solis R, Kalergis AM,Cespedes PF, Dudakovic A, Stein GS, van Wijnen AJ, Galindo M. ProteomicAnalysis of Exosomes and Exosome-Free Conditioned Media From HumanOsteosarcoma Cell Lines Reveals Secretion of Proteins Related to TumorProgression. J Cell Biochem. 2017;118:351–60.

51. Clark DJ, Fondrie WE, Yang A, Mao L. Triple SILAC quantitative proteomicanalysis reveals differential abundance of cell signaling proteins betweennormal and lung cancer-derived exosomes. J Proteomics. 2016;133:161–9.

52. Jingushi K, Uemura M, Ohnishi N, Nakata W, Fujita K, Naito T, Fujii R, SaichiN, Nonomura N, Tsujikawa K, Ueda K. Extracellular vesicles isolated fromhuman renal cell carcinoma tissues disrupt vascular endothelial cellmorphology via azurocidin. Int J Cancer. 2018;142:607–17.

53. Abramowicz A, Marczak L, Wojakowska A, Zapotoczny S, Whiteside TL, WidlakP, Pietrowska M. Harmonization of exosome isolation from culturesupernatants for optimized proteomics analysis. PLoS One. 2018;13:e0205496.

54. Hallal S, Mallawaaratchy DM, Wei H, Ebrahimkhani S, Stringer BW, Day BW,Boyd AW, Guillemin GJ, Buckland ME, Kaufman KL. Extracellular VesiclesReleased by Glioblastoma Cells Stimulate Normal Astrocytes to Acquire aTumor-Supportive Phenotype Via p53 and MYC Signaling Pathways. MolNeurobiol. 2019;56:4566–81.

55. Abramowicz A, Wojakowska A, Marczak L, Lysek-Gladysinska M, Smolarz M,Story MD, Polanska J, Widlak P, Pietrowska M. Ionizing radiation affects the

Kim et al. Proteome Science (2019) 17:5 Page 11 of 14

composition of the proteome of extracellular vesicles released by head-and-neck cancer cells in vitro. J Radiat Res. 2019;60(3):289-297.

56. Pan D, Chen J, Feng C, Wu W, Wang Y, Tong J, Zhou D. PreferentialLocalization of MUC1 Glycoprotein in Exosomes Secreted by Non-Small CellLung Carcinoma Cells. Int J Mol Sci. 2019;20(2):323.

57. Arbelaiz A, Azkargorta M, Krawczyk M, Santos-Laso A, Lapitz A, PerugorriaMJ, Erice O, Gonzalez E, Jimenez-Aguero R, Lacasta A, et al. Serumextracellular vesicles contain protein biomarkers for primary sclerosingcholangitis and cholangiocarcinoma. Hepatology. 2017;66:1125–43.

58. Clark DJ, Fondrie WE, Liao Z, Hanson PI, Fulton A, Mao L, Yang AJ.Redefining the Breast Cancer Exosome Proteome by Tandem Mass TagQuantitative Proteomics and Multivariate Cluster Analysis. Anal Chem. 2015;87:10462–9.

59. Atay S, Wilkey DW, Milhem M, Merchant M, Godwin AK. Insights into theProteome of Gastrointestinal Stromal Tumors-Derived Exosomes RevealsNew Potential Diagnostic Biomarkers. Mol Cell Proteomics. 2018;17:495–515.

60. Chen IH, Xue L, Hsu CC, Paez JS, Pan L, Andaluz H, Wendt MK, IliukAB, Zhu JK, Tao WA. Phosphoproteins in extracellular vesicles ascandidate markers for breast cancer. Proc Natl Acad Sci U S A. 2017;114:3175–80.

61. Wisniewski JR, Zougman A, Nagaraj N, Mann M. Universal samplepreparation method for proteome analysis. Nat Methods. 2009;6:359–62.

62. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin.2012;62:10–29.

63. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2019;69:7–34.64. Kruger S, Abd Elmageed ZY, Hawke DH, Worner PM, Jansen DA, Abdel-

Mageed AB, Alt EU, Izadpanah R. Molecular characterization of exosome-likevesicles from breast cancer cells. BMC Cancer. 2014;14:44.

65. Yamaguchi H, Condeelis J. Regulation of the actin cytoskeleton in cancercell migration and invasion. Biochim Biophys Acta. 1773;2007:642–52.

66. Kanojia D, Morshed RA, Zhang L, Miska JM, Qiao J, Kim JW, Pytel P,Balyasnikova IV, Lesniak MS. Ahmed AU: betaIII-Tubulin Regulates BreastCancer Metastases to the Brain. Mol Cancer Ther. 2015;14:1152–61.

67. Tang H, Wang Y, Zhang B, Xiong S, Liu L, Chen W, Tan G, Li H. High brainacid soluble protein 1(BASP1) is a poor prognostic factor for cervical cancerand promotes tumor growth. Cancer Cell Int. 2017;17:97.

68. Hsiao KC, Shih NY, Fang HL, Huang TS, Kuo CC, Chu PY, Hung YM, ChouSW, Yang YY, Chang GC, Liu KJ. Surface alpha-enolase promotesextracellular matrix degradation and tumor metastasis and represents a newtherapeutic target. PLoS One. 2013;8:e69354.

69. Tu SH, Chang CC, Chen CS, Tam KW, Wang YJ, Lee CH, Lin HW, Cheng TC,Huang CS, Chu JS, et al. Increased expression of enolase alpha in humanbreast cancer confers tamoxifen resistance in human breast cancer cells.Breast Cancer Res Treat. 2010;121:539–53.

70. Ding C, Fan X, Wu G. Peroxiredoxin 1 - an antioxidant enzyme in cancer. JCell Mol Med. 2017;21:193–202.

71. Bajor M, Zych AO, Graczyk-Jarzynka A, Muchowicz A, Firczuk M, Trzeciak L,Gaj P, Domagala A, Siernicka M, Zagozdzon A, et al. Targeting peroxiredoxin1 impairs growth of breast cancer cells and potently sensitises these cells toprooxidant agents. Br J Cancer. 2018;119:873–84.

72. Chen R, Kang R, Fan XG, Tang D. Release and activity of histone in diseases.Cell Death Dis. 2014;5:e1370.

73. Pemberton AD, Brown JK, Inglis NF. Proteomic identification of interactionsbetween histones and plasma proteins: implications for cytoprotection.Proteomics. 2010;10:1484–93.

74. Chen F, Wagner PD. 14-3-3 proteins bind to histone and affect both histonephosphorylation and dephosphorylation. FEBS Lett. 1994;347:128–32.

75. Nagai A, Sato T, Akimoto N, Ito A, Sumida M. Isolation and identification ofhistone H3 protein enriched in microvesicles secreted from culturedsebocytes. Endocrinology. 2005;146:2593–601.

76. Moon PG, Lee JE, Cho YE, Lee SJ, Chae YS, Jung JH, Kim IS, Park HY, BaekMC. Fibronectin on circulating extracellular vesicles as a liquid biopsy todetect breast cancer. Oncotarget. 2016;7:40189–99.

77. Purushothaman A, Bandari SK, Liu J, Mobley JA, Brown EE, Sanderson RD.Fibronectin on the Surface of Myeloma Cell-derived Exosomes MediatesExosome-Cell Interactions. J Biol Chem. 2016;291:1652–63.

78. Parente L, Solito E. Annexin 1: more than an anti-phospholipase protein.Inflamm Res. 2004;53:125–32.

79. Moraes LA, Ampomah PB, Lim LHK. Annexin A1 in inflammation and breastcancer: a new axis in the tumor microenvironment. Cell Adh Migr. 2018;12(5):417-423.

80. Okano M, Kumamoto K, Saito M, Onozawa H, Saito K, Abe N, Ohtake T,Takenoshita S. Upregulated Annexin A1 promotes cellular invasion in triple-negative breast cancer. Oncol Rep. 2015;33:1064–70.

81. Zhou X, Zhi X, Zhou P, Chen S, Zhao F, Shao Z, Ou Z, Yin L. Effects of ecto-5'-nucleotidase on human breast cancer cell growth in vitro and in vivo.Oncol Rep. 2007;17:1341–6.

82. Zhi X, Chen S, Zhou P, Shao Z, Wang L, Ou Z, Yin L. RNA interference ofecto-5'-nucleotidase (CD73) inhibits human breast cancer cell growth andinvasion. Clin Exp Metastasis. 2007;24:439–48.

83. Zhou P, Zhi X, Zhou T, Chen S, Li X, Wang L, Yin L, Shao Z, Ou Z.Overexpression of Ecto-5'-nucleotidase (CD73) promotes T-47D humanbreast cancer cells invasion and adhesion to extracellular matrix. CancerBiol Ther. 2007;6:426–31.

84. Wang L, Zhou X, Zhou T, Ma D, Chen S, Zhi X, Yin L, Shao Z, Ou Z, Zhou P.Ecto-5'-nucleotidase promotes invasion, migration and adhesion of humanbreast cancer cells. J Cancer Res Clin Oncol. 2008;134:365–72.

85. Jin D, Fan J, Wang L, Thompson LF, Liu A, Daniel BJ, Shin T, Curiel TJ, Zhang B.CD73 on tumor cells impairs antitumor T-cell responses: a novel mechanism oftumor-induced immune suppression. Cancer Res. 2010;70:2245–55.

86. Joosse SA, Hannemann J, Spotter J, Bauche A, Andreas A, Muller V, Pantel K.Changes in keratin expression during metastatic progression of breastcancer: impact on the detection of circulating tumor cells. Clin Cancer Res.2012;18:993–1003.

87. Dozier JH, Hiser L, Davis JA, Thomas NS, Tucci MA, Benghuzzi HA,Frankfurter A, Correia JJ, Lobert S. Beta class II tubulin predominatesin normal and tumor breast tissues. Breast Cancer Res. 2003;5:R157–69.

88. Palazzolo G, Albanese NN. G DIC, Gygax D, Vittorelli ML, Pucci-Minafra I:Proteomic analysis of exosome-like vesicles derived from breast cancer cells.Anticancer Res. 2012;32:847–60.

89. Moon PG, Lee JE, Cho YE, Lee SJ, Jung JH, Chae YS, Bae HI, Kim YB, Kim IS,Park HY, Baek MC. Identification of Developmental Endothelial Locus-1 onCirculating Extracellular Vesicles as a Novel Biomarker for Early Breast CancerDetection. Clin Cancer Res. 2016;22:1757–66.

90. Klinke DJ 2nd, Kulkarni YM, Wu Y, Byrne-Hoffman C. Inferring alterations incell-to-cell communication in HER2+ breast cancer using secretomeprofiling of three cell models. Biotechnol Bioeng. 2014;111:1853–63.

91. Cascone T, McKenzie JA, Mbofung RM, Punt S, Wang Z, Xu C, Williams LJ,Wang Z, Bristow CA, Carugo A, et al. Increased Tumor GlycolysisCharacterizes Immune Resistance to Adoptive T Cell Therapy. Cell Metab.2018;27:977–87 e974.

92. Ganapathy-Kanniappan S. Linking tumor glycolysis and immune evasion incancer: Emerging concepts and therapeutic opportunities. Biochim BiophysActa Rev Cancer. 1868;2017:212–20.

93. Gill KS, Fernandes P, O'Donovan TR, McKenna SL, Doddakula KK, PowerDG, Soden DM, Forde PF. Glycolysis inhibition as a cancer treatmentand its role in an anti-tumour immune response. Biochim Biophys Acta.1866;2016:87–105.

94. Leal AC, Mizurini DM, Gomes T, Rochael NC, Saraiva EM, Dias MS, WerneckCC, Sielski MS, Vicente CP, Monteiro RQ. Tumor-Derived Exosomes Inducethe Formation of Neutrophil Extracellular Traps: Implications For TheEstablishment of Cancer-Associated Thrombosis. Sci Rep. 2017;7:6438.

95. Khan S, Bennit HF, Turay D, Perez M, Mirshahidi S, Yuan Y, Wall NR. Earlydiagnostic value of survivin and its alternative splice variants in breastcancer. BMC Cancer. 2014;14:176.

96. Hannafon BN, Gin AL, Xu YF, Bruns M, Calloway CL, Ding WQ. Metastasis-associated protein 1 (MTA1) is transferred by exosomes and contributes tothe regulation of hypoxia and estrogen signaling in breast cancer cells. CellCommun Signal. 2019;17:13.

97. Yang SJ, Wang DD, Li J, Xu HZ, Shen HY, Chen X, Zhou SY, Zhong SL, ZhaoJH, Tang JH. Predictive role of GSTP1-containing exosomes inchemotherapy-resistant breast cancer. Gene. 2017;623:5–14.

98. Martinez VG, O'Neill S, Salimu J, Breslin S, Clayton A, Crown J, O'Driscoll L.Resistance to HER2-targeted anti-cancer drugs is associated with immuneevasion in cancer cells and their derived extracellular vesicles.Oncoimmunology. 2017;6:e1362530.

99. Wang T, Ning K, Lu TX, Sun X, Jin L, Qi X, Jin J, Hua D. Increasing circulatingexosomes-carrying TRPC5 predicts chemoresistance in metastatic breastcancer patients. Cancer Sci. 2017;108:448–54.

100. Ning K, Wang T, Sun X, Zhang P, Chen Y, Jin J, Hua D. UCH-L1-containingexosomes mediate chemotherapeutic resistance transfer in breast cancer. JSurg Oncol. 2017;115:932–40.

Kim et al. Proteome Science (2019) 17:5 Page 12 of 14

101. Steigedal TS, Toraskar J, Redvers RP, Valla M, Magnussen SN, Bofin AM,Opdahl S, Lundgren S, Eckhardt BL, Lamar JM. Nephronectin is correlatedwith poor prognosis in breast cancer and promotes metastasis via itsintegrin-binding motifs. Neoplasia. 2018;20:387–400.

102. Vardaki I, Ceder S, Rutishauser D, Baltatzis G, Foukakis T, Panaretakis T.Periostin is identified as a putative metastatic marker in breast cancer-derived exosomes. Oncotarget. 2016;7:74966.

103. Lakshmanan I, Ponnusamy MP, Macha MA, Haridas D, Majhi PD, Kaur S, JainM, Batra SK, Ganti AK. Mucins in lung cancer: diagnostic, prognostic, andtherapeutic implications. J Thorac Oncol. 2015;10:19–27.

104. Lakshmanan I, Rachagani S, Hauke R, Krishn SR, Paknikar S, Seshacharyulu P,Karmakar S, Nimmakayala RK, Kaushik G, Johansson SL, et al. MUC5ACinteractions with integrin beta4 enhances the migration of lung cancer cellsthrough FAK signaling. Oncogene. 2016;35:4112–21.

105. Cai F, Zhu Q, Miao Y, Shen S, Su X, Shi Y. Desmoglein-2 is overexpressed innon-small cell lung cancer tissues and its knockdown suppresses NSCLCgrowth by regulation of p27 and CDK2. J Cancer Res Clin Oncol.2017;143:59–69.

106. Al-Nedawi K, Meehan B, Micallef J, Lhotak V, May L, Guha A, Rak J.Intercellular transfer of the oncogenic receptor EGFRvIII by microvesiclesderived from tumour cells. Nat Cell Biol. 2008;10:619–24.

107. Chen W, Hoffmann AD, Liu H, Liu X. Organotropism: new insights intomolecular mechanisms of breast cancer metastasis. NPJ Precis Oncol.2018;2:4.

108. Li Z, Zeng C, Nong Q, Long F, Liu J, Mu Z, Chen B, Wu D, Wu H. ExosomalLeucine-Rich-Alpha2-Glycoprotein 1 Derived from Non-Small-Cell LungCancer Cells Promotes Angiogenesis via TGF-beta Signal Pathway. Mol TherOncolytics. 2019;14:313–22.

109. Li Y, Zhang Y, Qiu F, Qiu Z. Proteomic identification of exosomal LRG1: apotential urinary biomarker for detecting NSCLC. Electrophoresis.2011;32:1976–83.

110. Elahi S, Niki T, Hirashima M, Horton H. Galectin-9 binding to Tim-3 rendersactivated human CD4+ T cells less susceptible to HIV-1 infection. Blood.2012;119:4192–204.

111. Sandfeld-Paulsen B, Aggerholm-Pedersen N, Baek R, Jakobsen K, MeldgaardP, Folkersen B, Rasmussen T, Varming K, Jørgensen M, Sorensen B. Exosomalproteins as prognostic biomarkers in non-small cell lung cancer. Molecularoncology. 2016;10:1595–602.

112. Vykoukal J, Sun N, Aguilar-Bonavides C, Katayama H, Tanaka I, Fahrmann JF,Capello M, Fujimoto J, Aguilar M, Wistuba II. Plasma-derived extracellularvesicle proteins as a source of biomarkers for lung adenocarcinoma.Oncotarget. 2017;8:95466.

113. Jakobsen KR, Paulsen BS, Bæk R, Varming K, Sorensen BS, Jørgensen MM.Exosomal proteins as potential diagnostic markers in advanced non-smallcell lung carcinoma. Journal of extracellular vesicles. 2015;4:26659.

114. Huang SH, Li Y, Zhang J, Rong J, Ye S. Epidermal growth factor receptor-containing exosomes induce tumor-specific regulatory T cells. Cancer Invest.2013;31:330–5.

115. Jakobsen KR, Paulsen BS, Baek R, Varming K, Sorensen BS, Jorgensen MM.Exosomal proteins as potential diagnostic markers in advanced non-smallcell lung carcinoma. J Extracell Vesicles. 2015;4:26659.

116. Choi DS, Park JO, Jang SC, Yoon YJ, Jung JW, Choi DY, Kim JW, Kang JS,Park J, Hwang D, et al. Proteomic analysis of microvesicles derived fromhuman colorectal cancer ascites. Proteomics. 2011;11:2745–51.

117. Shiromizu T, Kume H, Ishida M, Adachi J, Kano M, Matsubara H, TomonagaT. Quantitation of putative colorectal cancer biomarker candidates in serumextracellular vesicles by targeted proteomics. Sci Rep. 2017;7:12782.

118. Ohshima K, Hatakeyama K, Kanto K, Ide T, Watanabe Y, Moromizato S,Wakabayashi-Nakao K, Sakura N, Yamaguchi K, Mochizuki T. Comparativeproteomic analysis identifies exosomal Eps8 protein as a potentialmetastatic biomarker for pancreatic cancer. Oncol Rep. 2019;41:1019–34.

119. Costa-Silva B, Aiello NM, Ocean AJ, Singh S, Zhang H, Thakur BK, Becker A,Hoshino A, Mark MT, Molina H, et al. Pancreatic cancer exosomes initiatepre-metastatic niche formation in the liver. Nat Cell Biol. 2015;17:816–26.

120. Madhavan B, Yue S, Galli U, Rana S, Gross W, Muller M, Giese NA, Kalthoff H,Becker T, Buchler MW, Zoller M. Combined evaluation of a panel of proteinand miRNA serum-exosome biomarkers for pancreatic cancer diagnosisincreases sensitivity and specificity. Int J Cancer. 2015;136:2616–27.

121. Raimondo F, Morosi L, Corbetta S, Chinello C, Brambilla P, Della Mina P, VillaA, Albo G, Battaglia C, Bosari S, et al. Differential protein profiling of renalcell carcinoma urinary exosomes. Mol Biosyst. 2013;9:1220–33.

122. Seyfried TN, Huysentruyt LC. On the origin of cancer metastasis. Crit RevOncog. 2013;18:43–73.

123. Jie XX, Zhang XY, Xu CJ. Epithelial-to-mesenchymal transition, circulatingtumor cells and cancer metastasis: Mechanisms and clinical applications.Oncotarget. 2017;8:81558–71.

124. Quiroz-Munoz M, Izadmehr S, Arumugam D, Wong B, Kirschenbaum A,Levine AC. Mechanisms of Osteoblastic Bone Metastasis in Prostate Cancer:Role of Prostatic Acid Phosphatase. J Endocr Soc. 2019;3:655–64.

125. Medeiros B, Allan AL. Molecular Mechanisms of Breast Cancer Metastasis tothe Lung: Clinical and Experimental Perspectives. Int J Mol Sci. 2019;20(9):2272.

126. Wang M, Zhao J, Zhang L, Wei F, Lian Y, Wu Y, Gong Z, Zhang S, Zhou J,Cao K, et al. Role of tumor microenvironment in tumorigenesis. J Cancer.2017;8:761–73.

127. Varol C. Tumorigenic Interplay Between Macrophages and CollagenousMatrix in the Tumor Microenvironment. Methods Mol Biol.1944;2019:203–20.

128. Segovia-Mendoza M, Morales-Montor J. Immune Tumor Microenvironmentin Breast Cancer and the Participation of Estrogens and Its Receptors IntoCancer Physiopathology. Front Immunol. 2019;10:348.

129. Giese MA, Hind LE, Huttenlocher A. Neutrophil plasticity in the tumormicroenvironment. Blood. 2019;133:2159–67.

130. Challagundla KB, Wise PM, Neviani P, Chava H, Murtadha M, Xu T, Kennedy R,Ivan C, Zhang X, Vannini I, et al. Exosome-mediated transfer of microRNAs withinthe tumor microenvironment and neuroblastoma resistance to chemotherapy. JNatl Cancer Inst. 2015;107(7). https://doi.org/10.1093/jnci/djv135.

131. Milane L, Singh A, Mattheolabakis G, Suresh M, Amiji MM. Exosomemediated communication within the tumor microenvironment. J ControlRelease. 2015;219:278–94.

132. Languino LR, Singh A, Prisco M, Inman GJ, Luginbuhl A, Curry JM, South AP.Exosome-mediated transfer from the tumor microenvironment increasesTGFbeta signaling in squamous cell carcinoma. Am J Transl Res.2016;8:2432–7.

133. Sun Z, Yang S, Zhou Q, Wang G, Song J, Li Z, Zhang Z, Xu J, Xia K, Chang Y,et al. Emerging role of exosome-derived long non-coding RNAs in tumormicroenvironment. Mol Cancer. 2018;17:82.

134. Hu C, Chen M, Jiang R, Guo Y, Wu M, Zhang X. Exosome-related tumormicroenvironment. J Cancer. 2018;9:3084–92.

135. Kalluri R. The biology and function of fibroblasts in cancer. Nat Rev Cancer.2016;16:582–98.

136. Hwang RF, Moore T, Arumugam T, Ramachandran V, Amos KD, Rivera A, JiB, Evans DB, Logsdon CD. Cancer-associated stromal fibroblasts promotepancreatic tumor progression. Cancer Res. 2008;68:918–26.

137. Xu Y, Lu Y, Song J, Dong B, Kong W, Xue W, Zhang J, Huang Y. Cancer-associated fibroblasts promote renal cell carcinoma progression. TumourBiol. 2015;36:3483–8.

138. Wei L, Ye H, Li G, Lu Y, Zhou Q, Zheng S, Lin Q, Liu Y, Li Z, Chen R. Cancer-associated fibroblasts promote progression and gemcitabine resistance viathe SDF-1/SATB-1 pathway in pancreatic cancer. Cell Death Dis. 2018;9:1065.

139. Silva TA, Smuczek B, Valadao IC, Dzik LM, Iglesia RP, Cruz MC, Zelanis A, deSiqueira AS, Serrano SM, Goldberg GS, et al. AHNAK enables mammarycarcinoma cells to produce extracellular vesicles that increase neighboringfibroblast cell motility. Oncotarget. 2016;7:49998–50016.

140. Takasugi M, Okada R, Takahashi A, Virya Chen D, Watanabe S, Hara E. Smallextracellular vesicles secreted from senescent cells promote cancer cellproliferation through EphA2. Nat Commun. 2017;8:15729.

141. Richards KE, Zeleniak AE, Fishel ML, Wu J, Littlepage LE, Hill R. Cancer-associated fibroblast exosomes regulate survival and proliferation ofpancreatic cancer cells. Oncogene. 2017;36:1770–8.

142. Ringuette Goulet C, Bernard G, Tremblay S, Chabaud S, Bolduc S, Pouliot F.Exosomes Induce Fibroblast Differentiation into Cancer-AssociatedFibroblasts through TGFbeta Signaling. Mol Cancer Res. 2018;16:1196–204.

143. Li W, Zhang X, Wang J, Li M, Cao C, Tan J, Ma D, Gao Q. TGFbeta1 infibroblasts-derived exosomes promotes epithelial-mesenchymal transition ofovarian cancer cells. Oncotarget. 2017;8:96035–47.

144. Wang L, Cao L, Wang H, Liu B, Zhang Q, Meng Z, Wu X, Zhou Q, Xu K.Cancer-associated fibroblasts enhance metastatic potential of lung cancercells through IL-6/STAT3 signaling pathway. Oncotarget. 2017;8:76116–28.

145. Hu Y, Yan C, Mu L, Huang K, Li X, Tao D, Wu Y, Qin J. Fibroblast-DerivedExosomes Contribute to Chemoresistance through Priming Cancer StemCells in Colorectal Cancer. PLoS One. 2015;10:e0125625.

Kim et al. Proteome Science (2019) 17:5 Page 13 of 14

146. Wen C, Seeger RC, Fabbri M, Wang L, Wayne AS, Jong AY. Biological rolesand potential applications of immune cell-derived extracellular vesicles. JExtracell Vesicles. 2017;6:1400370.

147. Jong AY, Wu CH, Li J, Sun J, Fabbri M, Wayne AS, Seeger RC. Large-scaleisolation and cytotoxicity of extracellular vesicles derived from activatedhuman natural killer cells. J Extracell Vesicles. 2017;6:1294368.

148. Lundholm M, Schroder M, Nagaeva O, Baranov V, Widmark A, Mincheva-Nilsson L, Wikstrom P. Prostate tumor-derived exosomes down-regulateNKG2D expression on natural killer cells and CD8+ T cells: mechanism ofimmune evasion. PLoS One. 2014;9:e108925.

149. Berchem G, Noman MZ, Bosseler M, Paggetti J, Baconnais S, Le Cam E,Nanbakhsh A, Moussay E, Mami-Chouaib F, Janji B, Chouaib S. Hypoxictumor-derived microvesicles negatively regulate NK cell function by amechanism involving TGF-beta and miR23a transfer. Oncoimmunology.2016;5:e1062968.

150. Xia Y, Zhang Q, Zhen Q, Zhao Y, Liu N, Li T, Hao Y, Zhang Y, Luo C, Wu X.Negative regulation of tumor-infiltrating NK cell in clear cell renal cellcarcinoma patients through the exosomal pathway. Oncotarget.2017;8:37783–95.

151. Zhu MC, Xiong P, Li GL, Zhu M. Could lung cancer exosomes induceapoptosis of natural killer cells through the p75NTR-proNGF-sortilin axis?Med Hypotheses. 2017;108:151–3.

152. Wang Y, Qin X, Zhu X, Chen W, Zhang J, Chen W. Oral cancer-derivedexosomal NAP1 enhances cytotoxicity of natural killer cells via the IRF-3pathway. Oral Oncol. 2018;76:34–41.

153. Abusamra AJ, Zhong Z, Zheng X, Li M, Ichim TE, Chin JL, Min WP. Tumorexosomes expressing Fas ligand mediate CD8+ T-cell apoptosis. Blood CellsMol Dis. 2005;35:169–73.

154. Huber V, Fais S, Iero M, Lugini L, Canese P, Squarcina P, Zaccheddu A,Colone M, Arancia G, Gentile M, et al. Human colorectal cancer cells induceT-cell death through release of proapoptotic microvesicles: role in immuneescape. Gastroenterology. 2005;128:1796–804.

155. Bergmann C, Strauss L, Wieckowski E, Czystowska M, Albers A, Wang Y,Zeidler R, Lang S, Whiteside TL. Tumor-derived microvesicles in sera ofpatients with head and neck cancer and their role in tumor progression.Head Neck. 2009;31:371–80.

156. Poggio M, Hu T, Pai CC, Chu B, Belair CD, Chang A, Montabana E, Lang UE,Fu Q, Fong L, Blelloch R. Suppression of Exosomal PD-L1 Induces SystemicAnti-tumor Immunity and Memory. Cell. 2019;177:414–27 e413.

157. Rong L, Li R, Li S, Luo R. Immunosuppression of breast cancer cellsmediated by transforming growth factor-beta in exosomes from cancercells. Oncol Lett. 2016;11:500–4.

158. Yamada N, Kuranaga Y, Kumazaki M, Shinohara H, Taniguchi K, Akao Y.Colorectal cancer cell-derived extracellular vesicles induce phenotypicalteration of T cells into tumor-growth supporting cells with transforminggrowth factor-beta1-mediated suppression. Oncotarget. 2016;7:27033–43.

159. Liu VC, Wong LY, Jang T, Shah AH, Park I, Yang X, Zhang Q, Lonning S,Teicher BA, Lee C. Tumor evasion of the immune system by convertingCD4+CD25- T cells into CD4+CD25+ T regulatory cells: role of tumor-derived TGF-beta. J Immunol. 2007;178:2883–92.

160. Chen W, Jiang J, Xia W, Huang J. Tumor-Related Exosomes Contribute toTumor-Promoting Microenvironment: An Immunological Perspective. JImmunol Res. 2017;2017:1073947.

161. Linden J, Cekic C. Regulation of lymphocyte function by adenosine.Arterioscler Thromb Vasc Biol. 2012;32:2097–103.

162. Deaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, Chen JF, EnjyojiK, Linden J, Oukka M, et al. Adenosine generation catalyzed by CD39 andCD73 expressed on regulatory T cells mediates immune suppression. J ExpMed. 2007;204:1257–65.

163. Cedeno-Laurent F, Dimitroff CJ. Galectin-1 research in T cell immunity: past,present and future. Clin Immunol. 2012;142:107–16.

164. Maybruck BT, Pfannenstiel LW, Diaz-Montero M, Gastman BR. Tumor-derivedexosomes induce CD8(+) T cell suppressors. J Immunother Cancer.2017;5:65.

165. Martinez FO, Gordon S. The M1 and M2 paradigm of macrophageactivation: time for reassessment. F1000Prime Rep. 2014;6:13.

166. Yang L, Zhang Y. Tumor-associated macrophages: from basic research toclinical application. J Hematol Oncol. 2017;10:58.

167. Baay M, Brouwer A, Pauwels P, Peeters M, Lardon F. Tumor cells and tumor-associated macrophages: secreted proteins as potential targets for therapy.Clin Dev Immunol. 2011;2011:565187.

168. Aras S, Zaidi MR. TAMeless traitors: macrophages in cancer progression andmetastasis. Br J Cancer. 2017;117:1583–91.

169. Chen Z, Yang L, Cui Y, Zhou Y, Yin X, Guo J, Zhang G, Wang T, He QY.Cytoskeleton-centric protein transportation by exosomes transforms tumor-favorable macrophages. Oncotarget. 2016;7:67387–402.

170. Zheng P, Luo Q, Wang W, Li J, Wang T, Wang P, Chen L, Zhang P, Chen H,Liu Y, et al. Tumor-associated macrophages-derived exosomes promote themigration of gastric cancer cells by transfer of functional Apolipoprotein E.Cell Death Dis. 2018;9:434.

171. Zhang X, Shi H, Yuan X, Jiang P, Qian H, Xu W. Tumor-derived exosomesinduce N2 polarization of neutrophils to promote gastric cancer cellmigration. Mol Cancer. 2018;17:146.

172. Wu L, Zhang X, Zhang B, Shi H, Yuan X, Sun Y, Pan Z, Qian H, Xu W.Exosomes derived from gastric cancer cells activate NF-kappaB pathway inmacrophages to promote cancer progression. Tumour Biol.2016;37:12169–80.

173. Chow A, Zhou W, Liu L, Fong MY, Champer J, Van Haute D, Chin AR, Ren X,Gugiu BG, Meng Z, et al. Macrophage immunomodulation by breast cancer-derived exosomes requires Toll-like receptor 2-mediated activation of NF-kappaB. Sci Rep. 2014;4:5750.

174. Piao YJ, Kim HS, Hwang EH, Woo J, Zhang M, Moon WK. Breast cancer cell-derived exosomes and macrophage polarization are associated with lymphnode metastasis. Oncotarget. 2018;9:7398–410.

175. Ham S, Lima LG, Chai EPZ, Muller A, Lobb RJ, Krumeich S, Wen SW,Wiegmans AP, Moller A. Breast Cancer-Derived Exosomes Alter MacrophagePolarization via gp130/STAT3 Signaling. Front Immunol. 2018;9:871.

176. Menck K, Klemm F, Gross JC, Pukrop T, Wenzel D, Binder C. Induction andtransport of Wnt 5a during macrophage-induced malignant invasion ismediated by two types of extracellular vesicles. Oncotarget. 2013;4:2057–66.

Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.

Kim et al. Proteome Science (2019) 17:5 Page 14 of 14